10408 Biochemistry 1991, 30, 10408-10415 Takesue, Y., Tamura, R., & Nishi, Y. (1977) Biochim. Bio- phys. Acta 483, 375-385. Toll, L., Brandt, S. R., Olsen, C. M., Judd, A. K., & Almquist, R. G. (1991) Biochem. Biophys. Res. Commun. 175, 886-893. Towbin, H., Staehelin, T., & Gordon, J. (1979) Proc. Natf. Wacker, H., Jaussi, R., Sonderegger, P., Dokow, M., Ghersa, P., Hauri, H.-P., Christen, Ph., & Semenza, G. (1981) FEBS Lett. 136, 329-332. Wong, J. T.-F. (1975) in Kinetics of Enzyme Mechanisms, pp 5-9, Academic Press Inc., New York. Yeh, K. Y., Yeh, M., & Holt, P. p. (1989) Am. J. Physiol. Acad. Sci. U.S.A. 76, 4350-4354. 256, G604-6 12. Molecular Basis for Vancomycin Resistance in Enterococcus faecium BM4 147: Biosynthesis of a Depsipeptide Peptidoglycan Precursor by Vancomycin Resistance Proteins VanH and VanAt Timothy D. H. Bugg,l Gerard D. Wright,* Sylvie Dutka-Malen,$ Michel Arthur,$ Patrice Courvalin,l and Christopher T. Walsh*v* Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 021 15, and Unit; des Agents Antibactgriens, Institut Pasteur, 28 Rue du Dr. Roux, 75724 Paris Cedex 15, France Received May 17, 1991; Revised Manuscript Received July 31, I991 ABSTRACT: Vancomycin resistance in Enterococcus faecium BM4 147 is mediated by vancomycin resistance proteins VanA and VanH. VanA is a D-alaninemalanine ligase of altered substrate specificity [Bugg, T. D. H., Dutka-Malen, S., Arthur, M., Courvalin, P., & Walsh, C. T. (1991) Biochemistry 30, 2017-20211, while the sequence of VanH is related to those of a-keto acid dehydrogenases [Arthur, M., Molinas, C., Dutka-Malen, S., & Courvalin, P. (1991) Gene (submitted)]. We report purification of VanH to homogeneity, characterization as a D-specific a-keto acid dehydrogenase, and comparison with D-lactate dehydrogenases from Leuconostoc mesenteroides and Lactobacillus leichmanii. VanA was found to catalyze ester bond formation between D-alanine and the D-hydroxy acid products of VanH, the best substrate being D-2- hydroxybutyrate (K, = 0.60 mM). The VanA product ~-alanyl-~-2-hydroxybutyrate could then be in- corporated into the UDPMurNAc-pentapeptide peptidoglycan precursor by D- Ala-D- Ala adding enzyme from Escherichia coli or by crude extract from E. faecium BM4147. The vancomycin binding constant of a synthetic modified peptidoglycan analogue N-acetyl-~-alanyl-~-2-hydroxybutyrate (& > 73 mM) was > 1 000-fold higher than the binding constant for N-acetyl-D-alanyl-D-alanine (& = 54 pM), partly due to the disruption of a hydrogen bond in the vancomycin-target complex, thus providing a molecular rationale for high-level vancomycin resistance. %e recent emergence of bacterial resistance to the glyco- peptide family of antibiotics in strains of Enterococcus faecium and Enterococcus faecalis (Courvalin, 1990) has prompted considerable interest in the mechanism of resistance: both because of the increasing use of the glycopeptide vancomycin in treatment of life-threatening Gram-positive bacterial in- fections and because of the unusual mode of action of the glycopeptide antibiotics. Vancomycin does not appear to penetrate the cell membrane or interact with cellular proteins but functions by complexation of peptidyl-DAla-D-Ala termini on the bacterial cell surface (Barna & Williams, 1984), thereby preventing transglycosylation and cross-linking of the peptidoglycan layer (Reynolds, 1989; Nagarajan, 1991). In view of the apparent ubiquity of the D-Ala-D-Ala terminus of bacterial peptidoglycan, it is difficult to envisage a simple mechanism for vancomycin resistance, which may explain the lack of bacterial resistance for over 30 years of clinical use. How then is this recently emergent vancomycin resistance achieved? 'Supported in part by NSF Grant DMB 8917290, a SERC/NATO postdoctoral fellowship (T.D.H.B.), and a NSERC postdoctoral fellow- ship (G.D.W.). * Author to whom correspondence should be addressed. * Harvard Medical School. 8 lnstitut Pasteur. 0006-2960/91/0430- 10408$02.50/0 High-level vancomycin resistance in E. faecium BM4 147, a class A vancomycin-resistant strain (Schlaes et al., 1991), is associated with production of a 38-kDa membrane-associ- ated protein VanA, whose amino acid sequence has been de- termined (Dutka-Malen et al., 1990). Sequence similarity was found with Gram-negative D-Ala-D-Ala ligases, which are cytoplasmic enzymes responsible for synthesis of the D-Ala- D-Ala dipeptide for peptidoglycan assembly (Walsh, 1989). VanA has been purified, and shows D-Ala-DAla ligase activity, but has substantially modified substrate specificity, compared with Gram-negative D-Ala-D-Ala ligases (Bugg et al., 1991). VanA is able to synthesize a number of mixed dipeptides including D-Ala-D-Met and D-Ala-D-Phe in preference to D-Ala-D-Ala, suggesting that its cellular role may be synthesis of a D-Ala-X dipeptide, which is incorporated into peptido- glycan and is able to be cross-linked but is not recognized by vancomycin. Sequencing of an open reading frame adjacent to the vanA gene on plasmid PIP816 that is also required for resistance has identified a further vancomycin resistance protein named VanH (Arthur et al., 1991). The amino acid sequence of VanH shows 19-30% sequence identity with sequences of three D-specific a-keto acid reductases: D-hydroxyisocaproate de- hydrogenase, D-3-phosphoglycerate dehydrogenase, and D- erythronate phosphate dehydrogenase. This raises the pos- 0 1991 American Chemical SocietyVancomycin Resistance Mechanism Biochemistry, Vol. 30, No. 43, 1991 10409 Table I: Purification of VanH from JM83/pSD8 purifica- vol (mL) act.‘ (unit/mL) proteinb (ma/mL) tot Drotein (mg) SD. act. (units/mn) tion (fold) crude extract 20.0 0.39 19.4 388 0.020 1 .o AcA54 gel filtration 48.0 0.28 1 .50 72 0.187 9.3 25-6076 (NHd)ZS04 7.5 0.91 26.0 195 0.037 1.86 2’.5’-ADP agarose 15.0 0.40 0.03 0.45 13.5 673 Mono-Q FPLC 7.5 0.61 0.009 0.0675 67.8 3,390 “Determined by NADH-dependent reduction of pyruvate at pH 5.6 (see Materials and Methods). One unit is defined as the activity required to convert 1 pmol of substrate to product per minute. bDetermined by Bradford protein assay (Bradford, 1976). sibility that VanH synthesizes a D-a-hydroxycarboxylic acid, which may be an alternative substrate for VanA. If VanA is able to